The Effect of 2-Ketobutyrate on Mitochondrial Substrate-Level Phosphorylation

Neurochemical Research (2019) 44:2301–2306 https://doi.org/10.1007/s11064-019-02759-8

ORIGINAL PAPER

David Bui1 · Dora Ravasz1 · Christos Chinopoulos1

Received: 13 December 2018 / Revised: 19 February 2019 / Accepted: 20 February 2019 / Published online: 27 February 2019 © The Author(s) 2019

Abstract The reaction catalyzed by succinate-CoA ligase in the mitochondrial matrix yields a high-energy phosphate when operating towards hydrolysis of the thioester bond of succinyl-CoA, known as mitochondrial substrate-level phosphorylation (mSLP). The catabolism of several metabolites converge to succinyl-CoA but through different biochemical pathways. Among them, threonine, serine and methionine catabolize to succinyl-CoA through the common intermediate, 2-ketobutyrate. During the course of this pathway 2-ketobutyrate will become succinyl-CoA through propionyl-CoA catabolism, obligatorily pass- ing through an ATP-consuming step substantiated by propionyl-CoA carboxylase. Here, by recording the directionality of the adenine nucleotide translocase while measuring membrane potential we tested the hypothesis that catabolism of 2-ketobutyrate negates mSLP due to the ATP-consuming propionyl-CoA carboxylase step in rotenone-treated, isolated mouse liver and brain mitochondria. 2-Ketobutyrate produced a less negative membrane potential compared to NADH or FADH2-linked substrates, which was sensitive to inhibition by rotenone, atpenin and arsenate, implying the involvement of complex I, complex II and a dehydrogenase—most likely branched chain keto-acid dehydrogenase, respectively. Co-addition of 2-ketobutyrate with NADH- or FADH2-linked substrates yielded no greater membrane potential than in the presence of substrates alone. However, in the presence of NADH-linked substrates, 2-ketobutyrate prevented mSLP in a dose-dependent manner. Our results imply that despite that 2-ketobutyrate leads to succinyl-CoA formation, obligatory metabolism through propionyl-CoA carboxylase associated with ATP expenditure abolishes mSLP. The provision of metabolites converging to 2-ketobutyrate may be a useful way for manipulating mSLP without using pharmacological or genetic tools.

Keywords Alpha-ketobutyrate · 2-Oxobutyrate · 2-Oxobutanoate · Succinyl-CoA

Introduction biochemical pathways encompassing at least one ATP- expenditure step, see Fig. 1. Catabolism of threonine and Mitochondrial substrate-level phosphorylation (mSLP) methionine converge to 2-ketobutyrate (2-KB, also known as mediated by succinate-CoA ligase is a reversible process α-ketobutyrate, 2-oxobutyrate, 2-oxobutanoate, CAS Regis- by which ATP (or GTP, depending on subunit composition try Number: 600-18-0), prior to entering into the propionate of the enzyme [1, 2]) can be generated in the absence of catabolic pathway towards succinyl-CoA. Serine joins the oxidative phosphorylation. This is feasible due to the high methionine catabolic pathway by combining with homocyst- energy stored in the thioester bond of succinyl-CoA. A num- eine forming cystathionine which forms 2-KB, cysteine and ber of metabolites converge towards succinyl-CoA; how- ammonia by cystathionine gamma-lyase. All of the reactions ever, with the exception of those catabolizing first through leading to 2-KB formation occur outside mitochondria, thus α-ketoglutarate, all others will obligatorily pass through 2-KB entry into the matrix for subsequent catabolism is war- ranted. Mindful that in the absence of oxidative phosphorylation the directionality of the adenine nucleotide translocase Special Issue: In honour of Prof. Vera Adam-Vizi. (ANT) and the reaction catalyzed by succinate-CoA ligase * Christos Chinopoulos are in directional synchrony [3] linked by the matrix [ATP]/ [email protected] [ADP] [4, 5], we hypothesized that metabolites converging to succinyl-CoA through ATP-consuming pathways would 1 Department of Medical Biochemistry, Semmelweis negate mSLP, and this would be reflected in the reversal of University, Tuzolto st. 37‑47, Budapest 1094, Hungary

Vol.:(0123456789)1 3 2302 Neurochemical Research (2019) 44:2301–2306

Fig. 1 Catabolism of metabolites towards succinyl-CoA. BCKDHC: branched-chain keto-acid dehydrogenase; BDH: β-hydroxybutyrate dehydrogenase; GLUD: glutamate dehydrogenase; GOT2: aspartate aminotransferase; KGDHC: ketoglutarate dehydrogenase complex; MCM: methylmalonyl mutase; MCEE: methylmalonyl racemase; NDPK: nucleoside diphosphokinase; PCC: propionyl-CoA carboxylase; SAM: S-adenosylmethionine; SDH: succinate dehydrogenase; SDS: l-serine dehydratase/l-threonine deaminase; SUCL: succinate- coA ligase. Dashed arrows imply multiple steps which may occur inside or outside the mitochondrial matrix. Entrance of 2-KB into the matrix likely occurs through the mitochondrial pyruvate carrier (depicted by a grey semi-transparent cylinder)

ANT when the electron transport chain is inhibited. Part of Determination of Membrane Potential in Isolated this work has been published before in abstract form [6]. Mitochondria

ΔΨm of isolated mitochondria (0.5 mg for mouse liver Materials and Methods and 0.25 mg for brain per 2 ml of medium) was estimated fluorimetrically with safranine O [9], acknowledging the Animals considerations elaborated in [10, 11] regarding inhibition of respiration as well as unspecific binding of safranine. Mice were of mixed 129 Sv and C57Bl/6 background. The Fluorescence was recorded in a Hitachi F-7000 spectro- animals used in our study were of either sex and between 2 fluorimeter (Hitachi High Technologies, Maidenhead, UK) and 6 months of age. Data obtained from liver mitochondria at a 5-Hz acquisition rate, using 495- and 585-nm excitation of mice of a particular gender or age (2, 4 or 6 months) and emission wavelengths, respectively. Experiments were did not yield any qualitative differences, thus all data were performed at 37 °C. pooled. Mice were housed in a room maintained at 20–22 °C Reagents: Standard laboratory chemicals and 2-ketobu- on a 12-h light–dark cycle with food and water available tyrate (Cat. No.: K401, purity 97%) were from Sigma. SF ad libitum. All experiments were approved by the Animal 6847 and atpenin A5 were from Enzo Life Sciences (ELS Care and Use Committee of the Semmelweis University AG, Lausen, Switzerland). Carboxyatractyloside (cATR) (Egyetemi Állatkísérleti Bizottság). was from Merck (Merck KGaA, Darmstadt, Germany). Mitochondrial substrate stock solutions were dissolved in Isolation of Mitochondria bi-distilled water and titrated to pH 7.0 with KOH. ADP was purchased as a K + salt of the highest purity available Liver and brain mitochondria were isolated as described in (Merck) and titrated to pH 6.9. Ref. [7]. Protein concentration was determined using the bicinchoninic acid assay, and calibrated using bovine serum standards [8] using a Tecan Infinite® 200 PRO series plate reader (Tecan Deutschland GmbH, Crailsheim, Germany).

1 3 Neurochemical Research (2019) 44:2301–2306 2303

Results

Catabolism of Metabolites Towards Succinyl‑CoA

As shown in Fig. 1, a number of metabolites converge to succinyl-CoA such as glutamine, threonine, serine, methionine, valine, isoleucine, thymine, cholesterol and of course others originating upstream from α-ketoglutarate. Dashed arrows imply multiple steps occurring in either inside or outside the mitochondrial matrix. Catabolism of threonine, serine and methionine lead to 2-KB generation which would enter the mitochondrial matrix and get converted to propionyl-CoA by the branched-chain keto-acid dehydrogenase complex (BCKDHC), and then subsequently to d-methylmalonyl-CoA by propionyl-CoA carboxylase (PCC), consuming ATP. In turn, d-methylmalonyl-CoA racemizes to l-methylmalonyl-CoA by methylmalonyl-CoA epimerase (MCEE) and then isomerizes to succinyl-CoA by methylmalonyl-CoA mutase (MCM), a B 12-dependent enzyme. The aforementioned enzymes reside inside the mitochondrial matrix, and since they process 2-KB it means that this metabolite traverses the inner Fig. 2 2-KB as a metabolic fuel in mouse liver mitochondria. Recon- mitochondrial membrane. To date, a 2-KB-specific carrier structed time courses of safranine O signal in isolated mouse liver has not been identified, though it is known to compete for mitochondria. The effect of 2-KB pulses (indicated by arrows signi- pyruvate transport through the mitochondrial pyruvate car- fying 0.5 mM each, thus a total of 3.5 mM 2-KB added) is shown. rier [12–14] and probably the newly identified choline car- Whenever indicated, succinate (5 mM) or rotenone (1 µM) or atpenin (atpn 1 µM), or arsenite (H 3AsO3, 1 mM) was added. At the end of rier [15, 16]. The reaction catalyzed by β-hydroxybutyrate each experiment 250 nM SF 6847 was added to achieve complete dehydrogenase (BDH) is also shown, demonstrating the depolarization (an increase in safranine O fluorescence signal implies competition between this enzyme complex and KGDHC depolarization). Wherever single graphs are presented, they are repre- for NAD +, in the presence of excess β-hydroxybutyrate. sentative of at least 4 independent experiments This phenomenon is exploited in our experimental settings in order to titrate the contribution of KGDHC yielding I inhibitor rotenone (rot, 1 µM) yielded no (liver mitochon- a-ketoglutarate in mitochondria with an inhibited respira- dria) or a very small depolarization implying intact opera- tory chain for the purpose of mSLP. From the above meta- tion of complexes III and IV. At the end of the experiment bolic considerations, we set to investigate if—and to what the uncoupler SF 6847 (SF, 250 nM) was added in order extent—2-KB serves as a fuel for mitochondria, and if so, to achieve a complete loss of ΔΨm indicating maximum does it impact on mSLP. safranine O fluorescence. However, addition of rotenone (Figs. 2b, 3b for liver and brain, respectively), or the complex II inhibitor atpenin (atpn, 1 µM, Figs. 2c, 3c for liver 2‑KB Supports Generation of ΔΨm in Isolated Liver and brain, respectively) or the dehydrogenases inhibitor and Brain Mitochondria by Generating Both NADH arsenite (H3AsO3 1 mM, Figs. 2d, 3d for liver and brain, and FADH2 respectively) abolished the effect of 2-KB conferring ΔΨm to mitochondria (with the exception of atpenin in brain As shown in Figs. 2 and 3 for liver and brain mito- mitochondria, where a mild decrease in safranine O fluo- chondria, respectively, addition of seven 0.5 mM 2-KB rescence was observed upon addition of 2-KB, Fig. 3c). pulses to mitochondria totaling 3.5 mM (panel Figs. 2a, As expected, subsequent addition of succinate (succ) led 3a) conferred a moderate decrease in safranine O fluo- to development of ΔΨm if mitochondria were challenged rescence implying development of ΔΨm. In liver mito- by rotenone or arsenite, but not atpenin. Note that prior to chondria, ΔΨm was afterwards becoming gradually lost, the addition of any substrates liver mitochondria exhibit as opposed to ΔΨm in brain mitochondria that remained an initial, transient polarization which is attributed to con- stable. Subsequent addition of succinate (succ, 5 mM) led sumption of endogenous substrates, most likely acyl car- to maximum polarization. Further addition of the complex nitines. This transient depolarization proceeds to a complete loss of ΔΨm within ~ 1 h. From the above results

1 3 2304 Neurochemical Research (2019) 44:2301–2306

Fig. 4 2-KB abolishes mSLP. Reconstructed time courses of safranine O signal in isolated mouse liver (a, b), or brain (c, d) mitochondria. Mitochondria were added where indicated by the closed circles. ADP (2 mM) was added where indicated. The effect of cATR (2 µM) Fig. 3 2-KB as a metabolic fuel in mouse brain mitochondria. Recon- on ΔΨm treated with rotenone (rot, 1 µM) in the absence or dose- structed time courses of safranine O signal in isolated mouse brain dependent presence of 2-KB as indicated in the legends is shown. mitochondria. The effect of 2-KB pulses (indicated by arrows signi- Control traces are shown in black. At the end of each experiment fying 0.5 mM each, thus a total of 3.5 mM 2-KB added) is shown. 250 nM SF 6847 was added to achieve complete depolarization (an Whenever indicated, succinate (5 mM) or rotenone (1 µM) or atpenin increase in safranine O fluorescence signal implies depolarization). (atpn 1 µM), or arsenite (H 3AsO3, 1 mM) was added. At the end of Wherever single graphs are presented, they are representative of at each experiment 250 nM SF 6847 was added to achieve complete least four independent experiments depolarization (an increase in safranine O fluorescence signal implies depolarization). Wherever single graphs are presented, they are representative of at least 4 independent experiments ATP synthase operation. Subsequently, oxidative phosphorylation was halted by inhibiting complex I with rotenone (rot). This led to a further loss of ΔΨm; ΔΨm was now we deduced that 2-KB affords ΔΨm to mitochondria by maintained by a reverse-function of Fo–F1 ATP synthase. mechanisms involving both complex I (fueled by NADH, Mindful of the directional synchrony of ANT with SUCL some originating from BCKDHC, an arsenite-sensitive reaction [4], Inhibition of the ANT by carboxyatractyloside enzyme complex) and complex II (fueled by FADH2). hints on the presence of mSLP: repolarization implies that SUCL operates towards ATP (or GTP) formation thus sup- 2‑KB Abolishes mSLP Conferred by Glutamate porting mSLP, while cATR-induced depolarization means or Pyruvate that SUCL was hydrolyzing ATP (or GTP). Substrates were either glutamate and malate (5 mM each) or glutamate and mSLP was addressed by interrogating the directionality of malate and 10 mM β-hydroxybutyrate (β-OH) (Fig. 4a, c) or the ANT in mitochondria with an inhibited respiratory chain pyruvate and malate (5 mM each) as indicated in the legends [3], isolated from mouse liver (Fig. 4a, b) or brain (Fig. 4c, of Fig. 4. Dose-dependent addition of 2-KB (0.5, 2 or 5 mM) d). The method is based on the electrogenic character of the converted the cATR-induced changes in safranine O fluo- ANT, as described in [3]; briefly, abolition of a “forward- rescence from a repolarization to a depolarization, imply- operating” ANT by carboxyatractyloside leads to a depo- ing dose-dependent inhibition of mSLP by 2-KB (which larization, while abolition of a reverse-operating ANT leads was complete at 5 mM, or 2 mM if β-hydroxybutyrate was to repolarization. As shown in Fig. 4, mitochondria were also present). This concentration range of 2-KB was chosen added where indicated by the closed circles and safranine for interrogating mSLP mindful of that used in Figs. 2, 3 O fluorescence was recorded reflecting ΔΨm. ADP (2 mM) verifying catabolism of this metabolite. At the end of each was added where indicated, initiating respiration and caus- experiment, the uncoupler SF 6847 SF, 250 nM was added ing a mild depolarization due to forward ANT and F o–F1 to achieve a completely depolarized state.

1 3 Neurochemical Research (2019) 44:2301–2306 2305

Discussion Relevant to this, it is important to emphasize that although ATP production mediated by succinyl-CoA ligase is small The most important observation of the present work is the compared to that produced by oxidative phosphorylation, abolition of mSLP by 2-KB in isolated mitochondria with mSLP assumes a critical role in preventing anoxic mito- an inhibited respiratory chain. This is likely attributed to chondria from becoming cytosolic ATP consumers, avoid- ATP expenditure by PCC, even though 2-KB supports suc- ing straining of glycolytic ATP reserves [3]. cinate-CoA ligase by providing succinyl-CoA. As a word Acknowledgements of caution though, the effect of BCKDHC stealingNAD + Open access funding provided by Semmelweis University (SE). We thank Dr. Maróthyné Tóth Erzsébet for helpful from KGDHC (when glutamate and malate were the main discussions. This work was supported by grants FIKP-61822-64888- fuels) could also contribute to abrogation of mSLP. The EATV and NKFIH KH129567 to C.C. decrease in NAD + provision to KGDHC with the aim of diminishing mSLP is the strategy followed by including Open Access This article is distributed under the terms of the Crea- β-hydroxybutyrate in the media (supporting NADH gen- tive Commons Attribution 4.0 International License (http://creativeco mmons.org/licen ses/by/4.0/ ), which permits unrestricted use, distribu- eration by β-hydroxybutyrate dehydrogenase) relying on tion, and reproduction in any medium, provided you give appropriate the fact that KGDHC activity is important for mSLP [7]. credit to the original author(s) and the source, provide a link to the Provision of NAD + to KGDHC in the absence of oxidative Creative Commons license, and indicate if changes were made. phosphorylation occurs through mitochondrially-localized diaphorases, [17] such as mitochondrially-localized NQO1 [18]. The effect of 2-KB inhibiting pyruvate oxidation has been published before [19], albeit this was only observed References at very high 2-KB concentrations (20 mM). This was attributed to a weak inhibitory action of 2-KB on the pyru- 1. Kacso G, Ravasz D, Doczi J, Nemeth B, Madgar O, Saada A, Ilin P, Miller C, Ostergaard E, Iordanov I, Adams D, Vargedo Z, vate dehydrogenase complex [20, 21]. Araki M, Araki K, Nakahara M, Ito H, Gal A, Molnar MJ, Nagy Our work identifies 2-KB (or metabolites converging Z, Patocs A, Adam-Vizi V, Chinopoulos C (2016) Two trans- towards this molecule) as abrogating mSLP in a physi- genic mouse models for beta-subunit components of succinate- ological manner. In human neutrophils, value ranges of CoA ligase yielding pleiotropic metabolic alterations. Biochem J 473:3463–3485 0.01–0.07 fmol/cell have been reported [22]; mindful 2. Johnson JD, Mehus JG, Tews K, Milavetz BI, Lambeth DO (1998) 3 that the volume of a human neutrophil is ~ 299 µm [23], Genetic evidence for the expression of ATP- and GTP-specific 2-KB cytosolic concentration must be 0.033–0.232 mM. In succinyl-CoA synthetases in multicellular eucaryotes. J Biol blood plasma and urine, 2-KB concentration is < 0.01 mM Chem 273:27580–27586 3. Chinopoulos C, Gerencser AA, Mandi M, Mathe K, Torocsik B, but can be elevated ten- or hundred-fold in certain disease Doczi J, Turiak L, Kiss G, Konrad C, Vajda S, Vereczki V, Oh states [24–26], thus, the flux of 2-KB production by sev- RJ, Adam-Vizi V (2010) Forward operation of adenine nucleotide eral metabolites may indeed reach a threshold upon which translocase during F0F1-ATPase reversal: critical role of matrix mSLP is affected. This can be exploited in a number of substrate-level phosphorylation. FASEB J 24:2405–2416 4. Chinopoulos C (2011) The “B Space” of mitochondrial phospho- metabolic settings in order to interrogate mSLP as part rylation. J Neurosci Res 89:1897–1904 of the citric acid cycle [27], further benefitted by it being 5. Chinopoulos C (2011) Mitochondrial consumption of cytosolic membrane-permeable. However, it must be emphasized ATP: not so fast. FEBS Lett 585:1255–1259 that 2-KB is also a substrate for lactate dehydrogenase 6. Bui D, Chinopoulos C (2018) The effect of 2-ketobutyrate on mitochondrial substrate level phosphorylation. Biochim Biophys being converted to α-hydroxybutyrate [28], thus, the effect Acta (BBA)-Bioenerg 1859:e100–e101 of 2-KB in whole cells or tissues may not only be attrib- 7. Kiss G, Konrad C, Doczi J, Starkov AA, Kawamata H, Manfredi uted to the mechanisms outlined in the present study. By G, Zhang SF, Gibson GE, Beal MF, Adam-Vizi V, Chinopoulos C the same token, the effects of 2-KB described in [28] could (2013) The negative impact of alpha-ketoglutarate dehydrogenase complex deficiency on matrix substrate-level phosphorylation. be partially ascribed to its ability of negating mSLP. FASEB J 27:2392–2406 Finally, since the catabolism of other metabolites con- 8. Smith PK, Krohn RI, Hermanson GT, Mallia AK, Gartner FH, verging to succinyl-CoA (except those originating from Provenzano MD, Fujimoto EK, Goeke NM, Olson BJ, Klenk DC α-Kg) besides threonine, serine and methionine also (1985) Measurement of protein using bicinchoninic acid. Anal- Biochem 150:76–85 require expenditure of one (such as in case of valine, iso- 9. Akerman KE, Wikstrom MK (1976) Safranine as a probe of the leucine, thymine) or two (such as with cholesterol) mol- mitochondrial membrane potential. FEBS Lett 68:191–197 ecules of ATP, it is expected that they will also lead to 10. Valle VG, Pereira-da-Silva L, Vercesi AE (1986) Undesirable fea- abolition of mSLP. The effect of catabolism of these latter ture of safranine as a probe for mitochondrial membrane potential. Biochem Biophys Res Commun 135:189–195 metabolites on mitochondrial ATP output with a dysfunc- 11. Chinopoulos C, Adam-Vizi V (2010) Mitochondrial Ca2 + tional respiratory chain is currently under investigation. sequestration and precipitation revisited. FEBS J 277:3637–3651

1 3 2306 Neurochemical Research (2019) 44:2301–2306

12. Bolli R, Nalecz KA, Azzi A (1989) Monocarboxylate and alpha- 22. Muhling J, Fuchs M, Campos ME, Gonter J, Engel JM, Sablotzki ketoglutarate carriers from bovine heart mitochondria. Purifica- A, Menges T, Weiss S, Dehne MG, Krull M, Hempelmann G tion by affinity chromatography on immobilized 2-cyano-4-hy- (2003) Quantitative determination of free intracellular alpha-keto droxycinnamate. J Biol Chem 264:18024–18030 acids in neutrophils. J Chromatogr B 789:383–392 13. Paradies G, Papa S (1975) The transport of monocarboxylic 23. Ting-Beall HP, Needham D, Hochmuth RM (1993) Volume and oxoacids in rat liver mitochondria. FEBS Lett 52:149–152 osmotic properties of human neutrophils. Blood 81:2774–2780 14. Hutson SM, Rannels SL (1985) Characterization of a mitochon- 24. Yang W, Roth KS (1985) Defect in alpha-ketobutyrate metabo- drial transport system for branched chain alpha-keto acids. J Biol lism: a new inborn error. Clin Chim Acta 145:173–182 Chem 260:14189–14193 25. Lee SH, Kim SO, Chung BC (1998) Gas chromatographic- 15. Michel V, Bakovic M (2009) The solute carrier 44A1 is a mito- mass spectrometric determination of urinary oxoacids using chondrial protein and mediates choline transport. FASEB J O-(2,3,4,5,6-pentafluorobenzyl)oxime-trimethylsilyl ester deri- 23:2749–2758 vatization and cation-exchange chromatography. J Chromatogr B 16. Jenden DJ (1978) Cholinergic mechanisms and psychopharmacol- 719:1–7 ogy. Springer, Boston 26. Bouatra S, Aziat F, Mandal R, Guo AC, Wilson MR, Knox C, 17. Kiss G, Konrad C, Pour-Ghaz I, Mansour JJ, Nemeth B, Starkov Bjorndahl TC, Krishnamurthy R, Saleem F, Liu P, Dame ZT, AA, Adam-Vizi V, Chinopoulos C (2014) Mitochondrial diapho- Poelzer J, Huynh J, Yallou FS, Psychogios N, Dong E, Bogumil rases as NAD(+) donors to segments of the citric acid cycle that R, Roehring C, Wishart DS (2013) The human urine metabolome. support substrate-level phosphorylation yielding ATP during res- PLoS ONE 8:e73076 piratory inhibition. FASEB J 28:1682–1697 27. Chinopoulos C (2013) Which way does the citric acid cycle turn 18. Ravasz D, Kacso G, Fodor V, Horvath K, Adam-Vizi V, Chi- during hypoxia? The critical role of alpha-ketoglutarate dehydro- nopoulos C (2018) Reduction of 2-methoxy-1,4-naphtoquinone genase complex. J Neurosci Res 91:1030–1043 by mitochondrially-localized Nqo1 yielding NAD(+) supports 28. Sullivan LB, Gui DY, Hosios AM, Bush LN, Freinkman E, Vander substrate-level phosphorylation during respiratory inhibition. Heiden MG (2015) Supporting aspartate biosynthesis is an essen- Biochim Biophys Acta Bioenerg 1859:909–924 tial function of respiration in proliferating cells. Cell 162:552–563 19. Gibson GE, Jope R, Blass JP (1975) Decreased synthesis of ace- tylcholine accompanying impaired oxidation of pyruvic acid in Publisher’s Note Springer Nature remains neutral with regard to rat brain minces. Biochem J 148:17–23 jurisdictional claims in published maps and institutional affiliations. 20. Kanzaki T, Hayakawa T, Hamada M, Fukuyoshi Y, Koike M (1969) Mammalian alpha-keto acid dehydrogenase complexes. IV. Substrate specificities and kinetic properties of the pig heart pyruvate and 2-oxyoglutarate dehydrogenase complexes. J Biol Chem 244:1183–1187 21. Blass JP, Lewis CA (1973) Kinetic properties of the partially purified pyruvate dehydrogenase complex of ox brain. Biochem J 131:31–37

1 3